A droplet actuator typically includes one or more substrates configured to form a surface or gap for conducting droplet operations. The one or more substrates establish a droplet operations surface or gap for conducting droplet operations and may also include electrodes arranged to conduct the droplet operations. The droplet operations substrate or the gap between the substrates may be coated or filled with a filler liquid that is immiscible with the liquid that forms the droplets.
Sequencing-by-synthesis (SBS) is a DNA sequencing strategy in which a template strand of DNA is used to synthesize its complement. Typically, SBS operates in a cyclical fashion wherein one or more nucleotide bases are added to a reaction containing the DNA templates, DNA polymerase, and other factors necessary to incorporate the next complementary bases into the synthesized strand. Depending on the specifics of the technology, a signal is then produced which enables one to infer the identity of the complementary bases incorporated into the synthesized strand. Although some approaches use a free-running enzyme to continuously synthesize a new strand, the majority of approaches currently in use require that the incorporation reactions remain synchronized at each cycle. This is typically achieved by either offering only one of the four nucleotide bases at a time or by using nucleotide bases that have reversible chemical blocks that prevent more than one base from being incorporated in any given cycle. Simple washing or reaction with de-blocking reagents is used between each cycle to prepare the DNA templates for the subsequent incorporation step. A single SBS experiment may incorporate 100s of these cycles leading to a need for very rapid and efficient liquid handling to cycle through the various liquid reagents used in each step.
Next-generation sequencing (NGS) systems employing SBS typically use flow-cells (FC) in which millions of these reactions are performed in parallel on a glass or silicon surface or upon the surfaces of microscopic beads. The FC serves to confine the reactions within a defined area where the signals can be conveniently detected, typically either by optical or electrical means. The liquid reagents are typically contained in tubes, plates, cartridges, or other disposable receptacles. A liquid-handling system (LHS) consisting of tubes, pumps, and valves is typically situated between the liquid reagents and the FC. The LHS conveys the liquids through the FC in a predetermined sequence dictated by the specific chemistry and the physical properties of the FC and LHS. Typically, the LHS utilizes valves to switch between the different liquid sources. These valves may be located some distance away from the FC. Thus a dead volume exists between each liquid source and the FC. This dead volume limits the efficiency of reagent utilization as well as the time required to switch between liquids. Therefore, there is a need for more efficient methods of liquid switching that are both more efficient in terms of reagent consumption and faster in terms of their ability to completely replace one liquid in the FC with a second liquid as the various steps in an NGS SBS protocol are performed.
Additionally, liquid-handling systems currently rely on the use of mechanical pumps and valves. Mechanisms with moving parts, such as these pumps and valves, are frequently found to be unreliable and difficult to maintain and operate. Therefore, there is a need for more simple liquid-handling systems that are less expensive, more reliable, and easier to operate.